Coupled Natural Fusion Enzymes in a Novel Biocatalytic Cascade Convert Fatty Acids to Amines

Tambjamine YP1 is a pyrrole-containing natural product. Analysis of the enzymes encoded in the Pseudoalteromonas tunicata “tam” biosynthetic gene cluster (BGC) identified a unique di-domain biocatalyst (PtTamH). Sequence and bioinformatic analysis predicts that PtTamH comprises an N-terminal, pyridoxal 5′-phosphate (PLP)-dependent transaminase (TA) domain fused to a NADH-dependent C-terminal thioester reductase (TR) domain. Spectroscopic and chemical analysis revealed that the TA domain binds PLP, utilizes l-Glu as an amine donor, accepts a range of fatty aldehydes (C7–C14 with a preference for C12), and produces the corresponding amines. The previously characterized PtTamA from the “tam” BGC is an ATP-dependent, di-domain enzyme comprising a class I adenylation domain fused to an acyl carrier protein (ACP). Since recombinant PtTamA catalyzes the activation and thioesterification of C12 acid to the holo-ACP domain, we hypothesized that C12 ACP is the natural substrate for PtTamH. PtTamA and PtTamH were successfully coupled together in a biocatalytic cascade that converts fatty acids (FAs) to amines in one pot. Moreover, a structural model of PtTamH provides insights into how the TA and TR domains are organized. This work not only characterizes the formation of the tambjamine YP1 tail but also suggests that PtTamA and PtTamH could be useful biocatalysts for FA to amine functional group conversion.


PtTamH Cloning and Expression Tests
PtTamH was cloned from Pseudoalteromonas tunicata D2 genomic DNA into pEHISTEV 1 using restriction digest and ligation cloning with the following primers: Expression of this PtTamH construct with a TEV-cleavable N-terminal 6 x His tag led to a large insoluble band, with very little soluble protein produced. The addition of sorbitol to the growth media as per literature 2 greatly increased protein solubility.

PtTamH Expression and Purification
Chemically-competent BL21 (DE3) cells were transformed using the pEHISTEV PtTamH expressing construct; successful transformants were screened on LB-agar plates supplemented with kanamycin (50 g mL -1 ). An overnight culture was subsequently prepared in kanamycin-LB media and incubated at 37 °C with agitation. A sample of the overnight culture was back-diluted to OD 600 = 0.1 in kanamycin-LB media supplemented with 500 mM sorbitol in 100 mM potassium phosphate, pH 7.5. At OD 600 = 0.6-0.8, protein expression was induced using 0.5 mM IPTG. The induced culture was incubated at 16 °C overnight with agitation. The biomass was harvested by centrifugation at 3500 xg for 15 minutes and stored at -20 °C until needed. The subsequent purification steps were carried out at 4 °C. The biomass was resuspended in lysis buffer (50 mM HEPES pH 8, 250 mM NaCl, 10 mM imidazole, 10 % glycerol, 25 µM PLP) and lysed by sonication for a total of 10 minutes (30 second pulse, 30 second cooldown). Cell debris was removed by centrifugation at 10,000 xg for 45 mins and the supernatant was clarified through a 0.45 micron filter. The cell-free extract was pumped through a 1 mL HiTrap TALON Crude column (Cytiva) using a 1 mL min -1 flow rate. The imidazole concentration was steadily increased remove any impurities before the protein was eluted with 250 mM imidazole. The fractions containing PtTamH were combined and dialysed with 1 mg polyhistidine-tagged TEV protease against the dialysis buffer (50 mM HEPES, 250 mM NaCl and 10 % glycerol) for 2 hrs. The dialysed protein mixture was pumped through a 1 mL HisTrap HP (Cytiva) to remove the TEV protease and any uncleaved PtTamH, and the untagged PtTamH was recovered in the flowthrough. Fractions containing untagged PtTamH were combined and the volume was reduced using a 30 kDa MWCO centrifugal concentrator. ~3.0 mg mL -1 of high-purity PtTamH could be routinely prepared using this IMAC strategy alone. For further characterisation, PtTamH was injected onto a 120 mL Superdex S200 (Cytiva) size exclusion column (SEC) pre-equilibrated with buffer (50 mM HEPES, 250 mM NaCl and 10 % glycerol). All purifications were monitored by SDS-PAGE.

PtTamH Aldehyde Reactions
Reactions contained 5 μM PtTamH, 250 μM PLP, 5 mM amine donor (ʟ -Glu or ʟ -Ala) and 1 mM C 6 -C 14 aldehyde (from a 10 mM stock in DMSO) in Buffer (50 mM HEPES, 100 mM NaCl and 1 mM DTT). The reactions were incubated at 37 °C for 24 hrs in triplicate and alongside control reactions prior to LC ESI-MS analysis.

Analysis of amine formation by LC ESI-MS
Amine reactions were quenched with a 1:1 volume of acetonitrile with 0.01% TFA and centrifuged at 17000 xg. 5 μL of supernatant was subjected to LC ESI-MS on a Synapt G2-Si Q-TOF (Waters) instrument with Phenomenex Jupiter C18 5 μm 300 Å LC column coupled to an ESI source. The LC gradient ran from 5% acetonitrile and 95% water with 0.1% formic acid to 95% acetonitrile over 30 min. The MS source was set at 120 °C, backing pressure 2 mbar and sampling cone voltage of 54 V. Extracted ion chromatograms (EICs) and masses were determined on MassLynx V4.1 software.

Detection of C 12 aldehyde by GC-MS
Reactions contained 5 μM PtTamH, 5 µM holo-PtTamA, 250 μM PLP, 2 mM NADH, 5 mM ATP and 1 mM C 12 acid (from a 25 mM stock in DMSO) in Buffer (50 mM HEPES, 200 mM KCl, 10 mM MgCl 2 and 1 mM DTT) in the absence of L-Glu amine donor. The reactions were incubated at 37 °C for 24 hrs in triplicate. The aldehyde product was extracted from the reaction mixture using an equivalent volume of EtOAc. The organic phase was sampled for GC-MS analysis using a Shimadzu QP2010 SE fitted with a Zebron ZB-FFAP capillary column (25 mm internal diameter [ID], 25 m film thickness, 30 m length). 1 L sample was injected (split 10:1 or 50:1, 230 °C inlet temperature using a Restek Topaz 3.5 mm ID quartz wool inlet liner) and chromatographically resolved under 1 mL min -1 constant helium flow using the following oven profile: 55 °C initial temperature (hold 2 minutes), 200 °C (20 °C min -1 ramp, hold 4 minutes), 240 °C (20 °C min -1 ramp, no hold). The MS was configured to detect ions over a range of 40-620 m/z following a 3.5 minute solvent delay. The ion source and transfer line temperatures were set to 180 °C.

Structure Prediction and Ligand Docking
All structural predictions were performed using ColabFold via AlphaFold2_advanced.ipynb. In brief, a deep multiple sequence alignment (MSA) was generated using MMSeqs2 prior to structure prediction using AlphaFold 2 (structural templates were not utilised for prediction). When appropriate, ColabFold was configured to perform homodimeric prediction. The output of the AlphaFold 2 structure module was recycled up to 6 times for refinement. For each sequence, a total of 5 models were generated and ranked by Predicted Template Model score (pTM); Predicted Local Distance Difference Test (pLDDT) scores were also computed for each model to evaluate fold-level confidence. The best model was subsequently relaxed to eliminate steric clashes. Visual inspection was performed in UCSF ChimeraX (v1.3) 3 and PyMOL (v2.4), and electrostatic potentials were computed using APBS Electrostatics. 4 Topological analysis was performed using the CASTp 3.0 server. 5 For docking studies, both the ligand and receptor were prepared using AutoDockTools 6 , and ligand docking was performed using AutoDock Vina (v1.1.2) 7, 8 . Ligand-receptor hydropathy surfaces were computed in BIOVIA Discovery Studio 2020.

Evolutionary Conservation Analysis
Evolutionary conservation analysis was performed using the ConSurf 13, 14, 15 server configured to build MSAs using MAFFT. 106 homologous sequences with identities ranging from 30-95% were compiled from UNIREF90 using the HMMER search algorithm. Conservation scores were calculated via the Bayesian method and visualised using UCSF ChimeraX 1.3.

Molecular Dynamics Simulation
Simulations were performed using GROMACS 2021.4. 9 Protein charges were computed using CHARMM36 all-atom forcefield. 10 The model was solvated in TIP3P water in a cubic box, and the net protein charge was counterbalanced using simulated sodium ions. The system was energy-minimised by sequential steepest descent/conjugate gradient descent and equilibrated to 300K and 1 bar using V-Rescale thermostat/Berendsen barostat. Following a 10 ns (5 x 10 6 time steps) production MD, the trajectory was re-centered with additional rotational and translational fitting. Further analysis was performed in GROMACS using gmx gyrate, gmx hbond and gmx rms. UCSF Chimera 1.16 11 was used for trajectory visualisation and for computing pairwise RMSDs.

Additional Notes on Predictive Modelling, Docking and Simulation:
Due to computational limitations, the PtTamH was modelled in three parts using the ColabFold parameters outlined in the Materials and Methods. First, the PtTamH ω-TA domain was accurately predicted as a homodimer (pLDDT: 90.27, pTM: 0.89); no plausible homomeric interface could be identified when docking the TR domain against itself (pTM < 0.6). Second, a separate prediction of the full PtTamH monomer was generated to gauge the relative orientation of the ω-TA and TR domains; the top-ranked output was predicted with high confidence (pLDDT: 92.27, pTM: 0.87, see Fig. 6B in main text). The final, complete homodimeric model was created by superimposition of two PtTamH monomers onto the predicted ω-TA dimer (RMSD: 0.395 Å between 510 pruned atom pairs). The structure was subsequently relaxed via a two-step steepest descent/conjugate gradient descent. Using AutoDock Vina and Vina forcefield, the C 12 -external aldimine was docked in the active site of the ω-TA domain with a calculated binding affinity of -7.6 kcal mol -1 . Similarly, both the C 12 aldehyde and NAD+ were docked in the TR domain with binding affinities of -3.5 kcal mol -1 and -10.1 kcal mol -1 respectively.
The complex was studied in a 10 ns (5 x 10 6 time steps) molecular dynamics simulation (MDS, see Methods and Materials). The interfacial contacts of the PtTamH ω-TA domains are maintained over the course of the MDS (see figure S18). In particular, the homomeric complex is stabilised by an average of 26 ± 5 hydrogen bonds, the majority of which (58%) occur within a distance of 2.67-2.93 Å. (figure S19A-B). While the average radius of gyration (R g = 4.88 ± 0.03 nm, R g max-min = 0.197 nm) suggests that the PtTamH complex is very stable, pairwise RMSD analysis reveals that the fusion enzyme may exhibit a moderate amount of conformational flexibility, with RMSDs as high as 6 Å occasionally observed (figure S19C-D). Throughout the MD trajectory, the TR domains remain oriented laterally from the ω-TA dimer interface with the putative substrate channel readily accessible for ACP docking. An accurate model of the PtTamA ACP was also predicted (pLDDT: 87.98), and complementary electrostatic surfaces between the ACP and PtTamH TR were identified using APBS electrostatics (see S20A-B). Figure S1: Tambjamine Biosynthetic pathway. A. Biosynthetic gene cluster (BGC) designated the 'tam' cluster responsible for the production of tambjamine YP1 in the Pseudoalteromonas tunicata organism. B. Predicted biosynthetic pathway for assembly of tambjamine YP1, shown as the production of the MBC core and free unsaturated amine which are then condensed together by TamQ to form the final molecule.    The MW of each protein is estimated using the following: